The alveolar and airway epithelium is recognized as a dynamic barrier that plays an important role in regulating inflammatory and metabolic responses to oxidative stress, sepsis, endotoxemia, and other critical illnesses in the lung. The respiratory epithelium, in particular, is a primary target of inflammatory conditions/infections at the epithelial-blood interface, and is itself capable of amplifying an inflammatory signal by recruiting inflammatory cells and producing inflammatory mediators.
Chronic Obstructive Pulmonary Disease (COPD) is one example of an inflammatory airway and alveolar disease where persistent upregulation of inflammation is thought to play a role. Inflammation in COPD is characterized by increased infiltration of neutrophils, CD8 positive lymphocytes, and macrophages into the airways. Neutrophils and macrophages play an important role in the pathogenesis of airway inflammation in COPD because of their ability to release a number of mediators including elastase, metalloproteases, and oxygen radicals that promote tissue inflammation and damage. It has been suggested that inflammatory cell accumulation in the airways of patients with COPD is driven by increased release of pro-inflammatory cytokines and of chemokines that attract the inflammatory cells into the airways, activate them and maintain their presence. The cells that are present also release enzymes (like metalloproteases) and oxygen radicals which have a negative effect on tissue and perpetuate the disease. A vast array of pro-inflammatory cytokines and chemokines have been shown to be increased within the lungs of patients with COPD. Among them, an important role is played by tumor necrosis factor alpha (TNF-alpha), granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 8 (IL-8), which are increased in the airways of patients with COPD.
Other examples of respiratory diseases where inflammation seems to play a role include: asthma, eosinophilic cough, bronchitis, acute and chronic rejection of lung allograft, sarcoidosis, pulmonary fibrosis, rhinitis and sinusitis. Asthma is defined by airway inflammation, reversible obstruction and airway hyperresponsiveness. In this disease the inflammatory cells that are involved are predominantly eosinophils, T lymphocytes and mast cells, although neutrophils and macrophages may also be important. A vast array of cytokines and chemokines have been shown to be increased in the airways and play a role in the pathophysiology of this disease by promoting inflammation, obstruction and hyperresponsiveness.
Eosinophilic cough is characterized by chronic cough and the presence of inflammatory cells, mostly eosinophils, within the airways of patients in the absence of airway obstruction or hyperresponsiveness. Several cytokines and chemokines are increased in this disease, although they are mostly eosinophil directed. Eosinophils are recruited and activated within the airways and potentially release enzymes and oxygen radicals that play a role in the perpetuation of inflammation and cough.
Acute bronchitis is an acute disease that occurs during an infection or irritating event for example by pollution, dust, gas or chemicals, of the lower airways. Chronic bronchitis is defined by the presence of cough and phlegm production on most days for at least 3 months of the year, for 2 years. One can also find during acute or chronic bronchitis within the airways inflammatory cells, mostly neutrophils, with a broad array of chemokines and cytokines. These mediators are thought to play a role in the inflammation, symptoms and mucus production that occur during these diseases.
Lung transplantation is performed in patients with end stage lung disease. Acute and more importantly chronic allograft rejection occur when the inflammatory cells of our body, lymphocytes, do not recognize the donor organ as “self”. Inflammatory cells are recruited by chemokines and cytokines and release a vast array of enzymes that lead to tissue destruction and in the case of chronic rejection a disease called bronchiolitis obliterans.
Sarcoidosis is a disease of unknown cause where chronic non-caseating granulomas occur within tissue. The lung is the organ most commonly affected. Lung bronchoalveolar lavage shows an increase in mostly lymphocytes, macrophages and sometimes neutrophils and eosinophils. These cells are also recruited and activated by cytokines and chemokines and are thought to be involved in the pathogenesis of the disease.
Pulmonary fibrosis is a disease of lung tissue characterized by progressive and chronic fibrosis (scarring) which will lead to chronic respiratory insufficiency. Different types and causes of pulmonary fibrosis exist but all are characterized by inflammatory cell influx and persistence, activation and proliferation of fibroblasts with collagen deposition in lung tissue. These events seem related to the release of cytokines and chemokines within lung tissue.
Acute rhinitis is an acute disease that occurs during an infection or irritating event, for example, by pollution, dust, gas or chemicals, of the nose or upper airways. Chronic rhinitis is defined by the presence of a constant chronic runny nose, nasal congestion, sneezing and pruritis. One can also find within the upper airways during acute or chronic rhinitis inflammatory cells with a broad array of chemokines and cytokines. These mediators are thought to play a role in the inflammation, symptoms and mucus production that occur during these diseases.
Acute sinusitis is an acute, usually infectious disease of the sinuses characterized by nasal congestion, runny, purulent phlegm, headache or sinus pain, with or without fever. Chronic sinusitis is defined by the persistence for more than 6 months of the symptoms of acute sinusitis. One can also find during acute or chronic sinusitis within the upper airways and sinuses inflammatory cells with a broad array of chemokines and cytokines. These mediators are thought to play a role in the inflammation, symptoms and phlegm production that occur during these diseases.
There is a growing body of evidence suggesting an intimate link between inflammation and neoplastic diseases. The tumor microenvironment is shaped by cells entering it, and their functions reflect the local conditions. Successive changes occurring at the tumor site during tumor progression resemble chronic inflammation. This chronic inflammatory reaction seems to be largely orchestrated by the tumor, and it seems to promote tumor survival. It has become evident that early and persistent inflammatory responses observed in or around developing neoplasms regulates many aspects of tumour development (matrix remodelling, angiogenesis, malignant potential) by providing diverse mediators implicated in maintaining tissue homeostasis, e.g., soluble growth and survival factors, matrix remodelling enzymes, reactive oxygen species and other bioactive molecules.
As described above, these inflammatory respiratory diseases or diseases in which inflammation plays a critical role are all characterized by the presence of mediators that recruit and activate different inflammatory cells which release enzymes or oxygen radicals causing symptoms, the persistence of inflammation and when chronic, destruction or disruption of normal tissue.
A logical therapeutic approach would be to downregulate cytokine and chemokine production and the inflammatory cell response. This has been performed in all the diseases described above by employing either topical or systemic corticosteroids with different levels of success. Corticosteroids are immune suppressive and have effects not only on inflammatory cells but also on other cells of the body that lead to toxicity when administered chronically.
Despite the availability of medications for COPD, asthma and other inflammatory respiratory diseases, the prevalence and morbidity of these diseases has remained stable or increased. It is obvious that there is an unmet medical need for the therapy of inflammatory respiratory diseases, and innovative therapeutic agents are urgently required. Antisense oligonucleotide-based therapy offers a new alternative approach to selectively decrease the expression of specific genes without the undesirable toxic effects of traditional therapeutic strategies. Antisense therapies are being investigated for the treatment of several diseases. It has been previously shown that antisense oligonucleotides directed against receptors for inflammatory mediators can be administered to the lungs and down-regulate their targets as described in WO9966037.
A therapeutic approach that would decrease pro-inflammatory cytokine and chemokine release by a vast array of cells while having a reduced effect on the release of anti-inflammatory mediators or enzymes may have an advantage over current therapies for inflammatory respiratory diseases or any other systemic inflammatory disease.
The cyclic nucleotides cAMP and cGMP are ubiquitous second messengers participating in signaling transduction pathways and mechanisms. Mammalian cells have evolved a complex and highly conserved complement of enzymes that regulate the generation and inactivation of cyclic nucleotides through multiple and complex feedforward and feedback mechanisms. Both cAMP and cGMP are formed from their respective triphosphates (ATP and GTP) by the catalytic activity of adenylyl (adenylate) or guanylyl (guanylate) cyclase, respectively as described in Essayan D. M. Cyclic nucleotide phosphodiesterase (PDE) inhibitors and immunomodulation. Biochem. Pharmacol., 1999, 57, 965-973. Inactivation of cAMP/cGMP is achieved by hydrolytic cleavage of the 3′-phosphodiester bond catalyzed by the cyclic-nucleotide-dependent phosphodiesterases (PDEs), resulting in the formation of the corresponding, inactive 5′-monophosphate as described in Essayan, 1999 and Perry M. J. and Higgs G. A. Chemotherapeutic potential of phosphodiesterase inhibitors. 1998, Curr Opin Chem Biol. 4:472-81.
It has been shown that the inflammatory response and its progression is exquisitely sensitive to modulations in the steady-state levels of cyclic nucleotides, where target cells for their effects extend beyond immune cells to include accessory cells, such as airway smooth muscle, epithelial and endothelial cells, and neurons as described in Perry and Higgs, 1998; Essayan, 1999. In this respect, the emerging concept that modulation of intracellular cyclic nucleotides plays a major role in regulating the inflammatory milieu has recently evolved into targeting and improving inflammatory/autoimmune responses. The cyclic nucleotide PDEs are a large, growing multigene family, comprising at least 11 families of PDE enzymes. The profile of selective and nonselective PDE inhibitors in vitro and in vivo, therefore, suggests a potential therapeutic utility as antidepressants, antiproliferative, immunomodulatory, tocolytics, inotropes/chronotropes, and cytoprotective agents.
Intracellular cAMP seems to have a fundamental role, not only in smooth muscle relaxation, activation and proliferation but also in the modulation of the release of mediators by inflammatory cells. Decreased cAMP levels can lead to increased production of inflammatory mediators such as TNF-alpha, GM-CSF, and IL-8 in airway epithelial cells.
Insight into the molecular mechanisms of the regulatory role of cytokines in cellular homeostasis as well as inflammatory/autoimmune/infectious diseases has begun to provide new approaches to design therapeutic strategies for pharmacological interventions. One such novel approach is the chemotherapeutic potential of PDE enzyme blockade, which revealed a phenomenal diversity and complexity scheme for promising therapeutics across a broad spectrum of disease states. One mechanistic understanding of PDE inhibition is centered on the immunomodulatory properties of cyclic nucleotides (cAMP/cGMP), thereby paving a channel through which anti-inflammatory, therapeutic applications could be clearly demonstrated.
The inflammatory response and its progression is exquisitely sensitive to modulations in the steady-state levels of cyclic nucleotides, where target cells for their effects extend beyond immune cells to include structural cells, such as epithelial, smooth muscle and endothelial cells, and neurons (Perry and Higgs, 1998; Essayan, 1999). Modulation of intracellular cyclic nucleotides plays a major role in regulating the inflammatory response. The cyclic nucleotide PDEs are a large, growing multigene family, comprising at least 11 families of PDE isoenzymes. PDEs differ in their tissue and cellular distribution as well as in their molecular and physicochemical characteristics including nucleotides and protein sequences, substrate specificity, inhibitor sensitivity, and cofactor requirements. Within different families, tissue specific isoforms are generated from the same gene by alternative mRNA splicing and differential promoter usage.
The cAMP-specific PDE4 enzyme family is one of the most extensively studied PDEs. Enzymes within this family are found in most pro-inflammatory and immune cells, where they play a key role in the regulation of cAMP metabolism. PDE4 enzymes are expressed in macrophages, neutrophils, cytotoxic CD8+ T cells, bronchial epithelial cells, and airway smooth muscle cells. Moreover, PDE4 inhibitors modulate inflammation in animal models of respiratory diseases, suggesting that PDE4 may represent a suitable target in a therapeutic based strategy for intervention with small molecule inhibitors. Anti-PDE4 drugs inhibit the hydrolysis of intracellular cAMP, which in turn provides bronchodilation and suppression of the inflammatory response. Selective PDE4 inhibitors such as cilomilast and roflumilast are active in animal models of neutrophil inflammation (Bundschuch D S et al. J. Pharmacol. Exp. Ther. 2001, 297: 280-290). Although the use of PDE4 inhibitors for the treatment of airway inflammation is under intensive clinical investigations, several inhibitors have been dropped because of their toxicity, dose-limiting side effects, of which nausea and vomiting are the most common physiological manifestations. Consequently, improving the therapeutic ratio of PDE4 inhibitors raised a major challenge that is still an important field of investigation.
Considering the distribution of enzymes in target tissues, with high activity of PDE3 and PDE4 in airway smooth muscle and inflammatory cells, selective inhibitors of these enzymes may add to the therapy of chronic airflow obstruction. Generally small molecule inhibitors have been focused on one or several PDE without assessing the potential detrimental effects of inhibiting all the isoenzymes of a PDE. For example it has been suggested that most of the toxicity attributed to the PDE4 antagonists will occur through inhibition of the PDE4 isotype D. In addition, as shown herein, inhibition of certain isoenzymes of PDEs does not decrease all pro-inflammatory mediators however, a combination of isotype specific oligonucleotides can lead to an effect that is much broader when the right combination of antisense oligonucleotides is employed.
The PDE3 family contains two different genes, PDE3A and PDE3B, which are cGMP-inhibited and display a high affinity towards cAMP. Each PDE3 gene codes for at least two splice isoforms. The PDE3A has been identified in smooth muscle, platelets and cardiac tissues. The PDE3B is most abundant in adipocytes and liver cells. However, initial data from clinical trials with selective PDE3 inhibitors or a combination with PDE4 inhibitors have been somewhat disappointing and have tempered the expectations considerably since these drugs had limited efficacy and their use was clinically limited through side effects.
PDE7 was first isolated from a human glioblastoma. PDE7A codes for a cAMP-specific PDE that is insensitive to cGMP and inhibitors of PDE3 and PDE4 and has an amino acid sequence distinct from other cAMP PDEs. In humans two genes (PDE7A and PDE7B) have been characterized. The PDE7A gene codes for three isoenzymes (PDE7A1, PDE7A2, and PDE7A3) derived from the same gene by alternative mRNA splicing. In humans, PDE7A2 mRNA is expressed abundantly in skeletal muscle, heart, and kidney, whereas the testis, lung, and immune system (thymus, spleen, lymph node, blood leukocytes) are rich sources of PDE7A1. In addition, activated, but not naïve, human T lymphocytes express the splice variant PDE7A3. In contrast, PDE7B exists as a single isoenzyme in humans, it shares ˜70% sequence similarity to PDE7A, but distinct kinetic properties. PDE7B is expressed predominantly in the brain and in a number of other tissues including liver, heart, thyroid glands, and skeletal muscle. Stimulation of human naïve T cells with anti-CD3 and anti-CD28 antibodies has been shown to promote IL-2 production and clonal amplification. These effects were attributed to PDE7A and down regulation of this enzyme prevents lymphocyte proliferation (Li L et al., Science, 1999, 283, 848-851).
The PDE4 family contains 4 different genes (PDE4 A-D). Due to alternative splicing of the genes, multiple splice variants are reported and classified into two main groups, the long and the short forms. PDE4A, B and D gene products are found in most immune and inflammatory cells. These are present either constitutively or after activation as described in Burnouf C. and Pruniaux M. P. Current Pharmaceutical Design 2002; 8:1255-1296. Inhibition of all or certain isotypes of PDE4 is associated with downregulation of several inflammatory mediators; however, an increase in certain pro-inflammatory cytokines (e.g. IL-6) has been described (Giembycz M. A. Expert Opin Invest Drugs 2001, 10:1361-1379).
It would therefore be desirable to inhibit all three PDE enzymes; however this approach may be plagued by the toxicity that has been described with administration of inhibitors of each of these enzymes. The topical application of antisense oligonucleotides could circumvent the systemic toxicity of enzyme inhibition but there appear to be too many isoenzymes of these PDE for this approach to be practical. Inhibition of one or several isotypes of PDE enzymes may not be as effective as full PDE inhibition since other isotypes would be present to have their pro-inflammatory effects.
It would therefore appear desirable to seek a way to down-regulate pro-inflammatory mediators and their cells while affecting less anti-inflammatory mediators or inhibitory enzymes for the therapy of inflammatory respiratory diseases. The therapeutic application of a combination of antisense oligonucleotides directed against selected isotypes of PDE enzymes is therefore herein proposed as a therapy for inflammatory respiratory diseases or any disease where increased cyclic AMP plays a role.